Female sperm storage in the bonnethead Sphyrna tiburo oviducal gland: Immunolocalization of steroid hormone receptors in sperm storage tubules

Highlights

• Hormonal regulation of female sperm storage was studied in bonnethead shark.

• Plasma levels of testosterone and 17β-estradiol increased during sperm storage.

• Plasma levels of progesterone increased during the end of the sperm storage period.

• Androgen, progesterone, and estrogen receptor α were found in sperm storage tubules.

• Estrogen receptor α was also detected in stored spermatozoa.

Abstract

Female sperm storage (FSS) has been demonstrated to occur in representatives from all major vertebrate groups and has been hypothesized to have several possible adaptive benefits that may maximize reproductive success of its practitioners. However, while the range of taxa that exhibit FSS and its possible evolutionary benefits have received significant attention in past years, the physiological mechanisms by which FSS occurs in vertebrates have only recently been explored. In this study, we examined the potential role of gonadal steroid hormones in regulating FSS in the bonnethead Sphyrna tiburo, a small hammerhead species in which females have been shown to be capable of storing male spermatozoa for up to 6 – 7 months following copulation. Like past studies on this species, we observed associations between plasma concentrations of the gonadal steroids 17β-estradiol, testosterone, and progesterone with FSS in female bonnetheads, suggesting roles for these hormones in regulating this process. Using immunohistochemistry, we also observed presence of androgen receptor, estrogen receptor alpha (ERα), and progesterone receptor in epithelial cells of sperm storage tubules in the bonnethead oviducal gland, as well as occurrence of ERα in stored spermatozoa, specifically during the sperm storage period. These results suggest that E₂, T, and P₄ may regulate certain aspects of FSS in bonnethead indirectly through actions on the female reproductive tract, whereas E₂ may also have direct effects on sperm function. This is the first study on the regulation of FSS in sharks and has formed a basis for future work geared towards improving our understanding of this process in chondrichthyans.

Introduction

The prolonged occurrence and survival of male spermatozoa in the female reproductive tract following copulation, the process generally referred to as female sperm storage (FSS), has been reported to occur in representatives from all major vertebrate groups including fish, amphibians, reptiles, birds, and mammals (Birkhead and Møller, 1993, Holt and Lloyd, 2010, Holt and Fazeli, 2016). FSS is believed to have evolved independently multiple times among the vertebrates and has been linked with several adaptive benefits, perhaps explaining its broad distribution among vertebrate groups (Matsuzaki and Sasanami, 2017). By providing a temporal separation between insemination and fertilization that can range from hours in some mammals to possibly up to 7 years in some reptiles, FSS may make it possible for females to coordinate certain, perhaps vulnerable stages of the reproductive cycle during optimal environmental and/or physiological conditions (Holt and Fazeli, 2016). The practice of FSS may also maximize reproductive success in species that exhibit asynchronous male and female reproductive cycles, as well as those that experience sexual segregation and show only limited spatial overlap between males and females (Birkhead and Møller, 1993, Ortega-León et al., 2009). FSS has also been hypothesized to provide a mechanism for post-copulatory forms of cryptic female choice, an adaptation that may allow females to bias fertilization success towards gametes from genetically favorable males, thereby enhancing offspring success (Pearse and Avise, 2001, Hemmings and Birkhead, 2017).

Recently, there has been a notable increase in studies that have explored the still poorly understood physiological mechanisms by which FSS occurs in vertebrates (Holt and Fazeli, 2016, Matsuzaki and Sasanami, 2017). Although these processes are likely to be as diverse as the sites within the female reproductive tract in which sperm storage has been reported to occur in, some commonalities have been observed, even in highly divergent taxa. It has been demonstrated in several species that spermatozoa likely interact with epithelial cells in sperm storage organs following insemination to induce changes that likely result in producing a microenvironment conducive to the prolonged survival of spermatozoa (Holt and Fazeli, 2016). This may include changes in epithelial cell-production of factors that reduce the potential for oxidative stress and/or apoptosis in spermatozoa, as well as those that suppress the immune response in the female reproductive tract. The female endocrine system is expected to play a key role in regulating such processes through direct actions on cells in sperm storage organs and/or perhaps spermatozoa themselves. For example, plasma concentrations of androgens rise specifically during the sperm storage period in females of both the Chinese soft-shelled turtle Pelodiscus sinensis (Liu et al., 2016) and the greater Asiatic yellow bat Scotophilus heathi (Roy and Krishna, 2010), and receptors for these hormones are present in sites of sperm storage in both species (Roy and Krishna, 2010, Roy and Krishna, 2011, Liu et al., 2016). In S. heathi, androgens appear to promote the survival of stored spermatozoa by regulating the balance of anti- versus pro-cell survival factors produced by sperm storage organs (Roy and Krishna, 2011). Both estrogens and progestins have also been implicated in regulating certain aspects of FSS in various vertebrates (Yoshimura et al., 2000, Das et al., 2006, Madekurozwa et al., 2009, Foye-Jackson et al., 2011, Ito et al., 2011, Riou et al., 2016, Khillare et al., 2018) with progestins specifically linked with the release of spermatozoa from sperm storage reservoirs, perhaps through effects on sperm motility and/or adhesion between spermatozoa and epithelial cells in the female reproductive tract (Machado et al., 2019, Romero-Aguirregomezcorta et al., 2019, Ramal-Sanchez et al., 2020).

In the cartilaginous fishes (Class Chondrichthyes: sharks, skates, rays, and chimaeras), FSS is generally believed to occur in epithelial cell-lined sperm storage tubules (SSTs) of the oviducal glands (also known as shell glands or nidamental glands), enlarged portions of the anterior oviducts that are also responsible for the production of tough or flexible egg envelopes (depending on the reproductive mode of the species in question) and various jelly coats and additional egg investments that lie between the egg envelope and the fertilized ovum (Hamlett et al., 2005). Although numerous studies have described presence of spermatozoa in this organ in various chondrichthyans (Pratt, 1993, Hamlett et al., 1998, Conrath et al., 2002, Hamlett et al., 2002a, Hamlett et al., 2002b, Galíndez and Estecondo, 2008, Storrie et al., 2008, Moura et al., 2011, Serra-Pereira et al., 2012, Porcu et al., 2015, Elías, 2015a, Elías, 2015b, Marongiu et al., 2015, Rangel et al., 2015, Maduna et al., 2018, Soto-López et al., 2018, Gonzalez De Acevedo et al., 2020), no published studies to date have explored the physiological mechanisms by which FSS occurs in these fishes. Notwithstanding this point, studies on the bonnethead Sphyrna tiburo have suggested that gonadal steroid hormones may regulate FSS in cartilaginous fishes in manners similar to those described for other vertebrates, based on associations between circulating concentrations of these hormones and the sperm storage period. In particular, it has been demonstrated that plasma concentrations of both 17β-estradiol (E₂) and testosterone (T) increase significantly in post-copulatory/pre-ovulatory female bonnetheads during the sperm storage period, whereas progesterone (P₄) levels exhibit a transient rise during the end of the sperm storage period and ovulation (Manire et al., 1995, Gonzalez De Acevedo et al., 2020). However, due to the lack of direct evidence for the presence of gonadal steroid receptors in bonnethead SSTs, these data are at best correlative and require further investigation.

The goal of this study was therefore to determine if SSTs in the chondrichthyan oviducal gland are targets for the actions of gonadal steroid hormones. To accomplish this, we examined the presence and distribution of cellular receptors for androgens (AR), estrogens (ER), and progestins (PR) in SSTs and other components of the oviducal gland of the bonnethead. As previously described, this species exhibits a well-characterized period of FSS that has been linked with temporal changes in circulating levels of T, E₂, and P₄ (Manire et al., 1995, Gonzalez De Acevedo et al., 2020). Studies on reproduction in both northern Gulf of Mexico and northwest Atlantic bonnetheads show that male spermatogenesis occurs from late spring to late summer annually, followed by copulation between fall and early winter (Parsons, 1993, Manire et al., 1995, Gelsleichter et al., 2002, Gelsleichter et al., 2003, Gonzalez De Acevedo et al., 2020). Depending on the population in question, females store spermatozoa for 4 – 7 months afterwards in oviducal gland SSTs until ovulation and fertilization take place during the subsequent spring (Manire et al., 1995, Gonzalez De Acevedo et al., 2020). Parturition generally occurs in late summer – early fall after a 4.5- to 5-month gestation period, the shortest reported in any shark species (Parsons, 1993, Manire et al., 1995, Manire and Rasmussen, 1997, Lombardi-Carlson et al., 2003, Nichols et al., 2003, Gonzalez De Acevedo et al., 2020). We specifically examined gonadal steroid receptor localization in the oviducal gland of northwest Atlantic bonnetheads, which have been demonstrated to copulate in late September and ovulate near the end of the following April; a 7-month period of sperm storage (Gonzalez De Acevedo et al., 2020). In addition to providing new information on the regulation of FSS in vertebrates, this study was also motivated by a need to better understand how the natural abilities of some female chondrichthyans to maintain sperm vitality over prolonged periods can be harnessed for use in captive animal breeding programs. As recently suggested by Wyffels et al. (2020), this may enable aquaria to maintain self-sustaining populations of sharks and rays under managed care, reducing their reliance on increasingly threatened natural populations.