Seminal quality and global proteomic analysis of spermatozoa from captive Amazon squirrel monkeys (Saimiri collinsi Osgood, 1916) during the dry and rainy seasons

The squirrel monkey (Saimiri collinsi), a Neotropical primate endemic to the Amazon in Brazil, is used as a biological model for reproductive research on the genus Saimiri. Although this animal is known to exhibit reproductive seasonality, nothing is known about the differences in its seminal quality, sperm protein composition, or sperm protein profile between the breeding (dry) and non-breeding (rainy) seasons. Thus, the aims of this study were to evaluate the quality of S. collinsi semen during the dry and rainy seasons and to describe the global sperm proteomics and expression variations in the sperm proteins during the two seasons. Aside from the pH, there was no difference in the seminal quality between the dry and rainy seasons. The study approach based on bottom-up proteomics allowed the identification of 2343 proteins present in the sperm samples throughout these two seasons. Of the 79 proteins that were differentially expressed between the two seasons, 39 proteins that were related to spermatogenesis, sperm motility, capacitation, fecundation, and defense systems against oxidative stress were upregulated in the dry season. Knowledge on the sperm proteins provides crucial information for elucidating the underlying mechanisms associated with sperm functionality. Thus, our results help to advance our understanding of the reproductive physiology of S. collinsi, providing valuable information for the improvement of protocols used in assisted reproduction techniques for the conservation of endangered Saimiri species.


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The squirrel monkey (Saimiri collinsi), a Neotropical primate endemic to the Amazon in 45 Brazil [1], is commonly used as an experimental model for reproductive research on the genus 46 Saimiri [2][3][4]. According to the International Union for Conservation of Nature's Red List of 47 Threatened Species, two Saimiri species are ranked as vulnerable (Saimiri oerstedii and Saimiri 48 vanzolini) and one species as almost threatened (Saimiri ustus) to extinction [5]. 49 Primates of the genus Saimiri exhibit reproductive seasonality. In the free-living animals, the 50 breeding season (mating) and births occur during the dry season and rainy season, respectively. 51 Supposedly, the rainy season is when there is more food available for the newborn [6][7][8]. However, 52 Saimiri monkeys that are held in captivity without variations in their environment and food supply 53 express less of a seasonality pattern by continuing to mate and reproduce throughout the year [9]. 54 Because of the conflicting observations between free-range and captive individuals, it is obvious 55 that the effects of environmental factors (e.g., rainfall, temperature, photoperiod, and food supply) 56 on reproductive seasonality need to be more fully understood [9][10][11]. 57 Although studies on the squirrel monkey have already shown correlations between 58 reproductive seasonality and spermatogenesis (Saimiri sciureus) [12] and the gonadal hormones (S. 59 sciureus) [13], only one study has reported the seasonal influence on seminal quality (S. sciureus) 60 [14]. However, nothing is known about the protein composition of spermatozoa in these Neotropical 61 primates, or of the differences in the sperm protein profile between the breeding and non-breeding 62 seasons. In domestic animals, proteomic studies have shown the upregulation and downregulation of 63 expression of some sperm proteins when the breeding and non-breeding seasons are compared [15]. 4 64 Mammalian male fertility depends on physiological events that begin with spermatogenesis 65 and culminate with successful adhesion/signaling between the sperm membrane and the 66 extracellular coat of the oocyte, followed by adhesion/fusion between the oocyte and sperm 67 membranes during fertilization in the female reproductive tract [16,17]. Proteins expressed by 68 spermatozoa and those from the seminal plasma that bind to the sperm plasma membrane render the 69 spermatozoa capable of fertilizing a mature oocyte [18,19]. Studies in animals and humans have 70 described sperm proteins that have significant associations with sperm motility (i.e., L-lactate 71 dehydrogenase and dynein heavy chain 1 (DNAH1)) [20, 21], sperm capacitation (i.e., clusterin, 72 spermadhesin, and mitochondrial peroxiredoxin-5) [22,23], and fertility (i.e., enolase 1, ropporin- 73 1-like protein (ROPN1), and Izumo sperm-egg fusion 1 (IZUMO1)) [24,25]. 74 In non-human primates, sperm proteomics has been carried out only in Old World primates 75 for characterization of the sperm protein profile [18,[26][27][28][29]. Although these studies have been 76 carried out in the genus Macaca, which also exhibits reproductive seasonality [30], nothing is 77 known about the changes that may occur in the sperm protein profile during the non-breeding and 78 breeding seasons, and the influence of these changes on the seminal quality of these animals. 79 Knowledge about the absence, presence, underexpression, or overexpression of these sperm proteins 80 could help to further our understanding of the mechanisms behind the reduction in the fertilization 81 ability of sperm [19,31]. 82 Defining the sperm protein profiles of Saimiri collinsi in the breeding (dry season) and non-83 breeding (rain season) seasons may provide us with a better understanding about the reproductive 84 physiology of these animals, as well as whether the sperm cells could be used in assisted 85 reproduction techniques throughout the year rather than being restricted only to the breeding period. fruits, vegetables, commercial pellet chow specific for Neotropical non-human primates (18% 125 protein, 6.5% fiber; Megazoo, Minas Gerais, Brazil), and cricket larvae (Zophobas morio). 126 Vitamins, minerals, and eggs were supplied once a week, and water was available ad libitum. König S.A.) and monitored by a veterinarian. After anesthesia, the animals were weighed using a 135 weight balance, and the testicular length, width, height, and circumference were measured using a 136 universal caliper. The testicular volume was calculated according to the method described by 137 Oliveira et al. [4]. After the animal had been placed in dorsal recumbency, the genital region was 138 sanitized with a mild soap and distilled water (1:10) and the prepuce was retracted for a more   The sperm motility, vigor, and morphology were evaluated according to the methods 157 described by Oliveira et al. [2][3][4]. For evaluation of the normal sperm morphology and plasma 158 membrane integrity, a smear sample was prepared by adding 5 µL of 1% eosin (Vetec, Rio de 159 Janeiro, Brazil) and 5 µL of 1% nigrosine (Vetec, Rio de Janeiro, Brazil) to 5 µL of semen on a

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Characteristics of the Amazon monkeys and semen 236 The body weights of the male monkeys and their total testicular volumes were significantly 237 higher in the rainy season (883.15 ± 14.50 g and 2.42 ± 0.11 cm 3 , respectively) than in the dry 238 season (816.10 ± 6.85 g and 1.91 ± 0.13 cm 3 ) ( Table 1) were whitish, 57% were yellowish, 46% were transparent, and 54% were opaque. There was a 243 statistical difference (p = 0.0002) in the seminal pH between the dry (7.96 ± 0.10) and rainy (7.30 ± year (dry or rainy season) ( Table 1). The study approach based on bottom-up proteomics allowed the identification of 2343 258 proteins in the sperm samples (Supporting Information S1 Table). Of the total proteins identified, 259 223 were determined to participate in important reproductive events, such as spermatogenesis (67 260 proteins), sperm motility (42 proteins), capacitation/acrosome reaction (20 proteins), and 261 fertilization (32 proteins) (Supporting Information S2 Table).

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On the basis of the GO analysis, the proteins were grouped according to biological process, 263 molecular function, and cellular component (i.e., localization) classes (Fig 1). In the cellular 264 component class, most of the proteins identified were associated with the cytoplasm (12.3%), 265 cytoskeleton (9.4%), and nucleus (8.9%) (Fig 1A). The most common biological processes 266 associated with the proteins were cellular processes (41.6%), regulation (17.6%), and metabolic 267 processes (11.4%) (Fig 1B). Binding (42.8%) and catalytic activity (42.9%) corresponded to the 268 most frequent molecular functions for the proteins (Fig 1C).  and matrix metalloproteinase-7), cellular regulation (i.e., amine oxidase and serine protease 30-like), 278 and immune system processes (i.e., heat shock 70 kDa protein 1A/1B and clusterin) ( Table 2 and   279 Supporting Information S3 Table). With regard to proteins that participate in important events in 280 reproduction, 10 that were increased during the dry season were related to spermatogenesis (i.e., cat  In silico protein network analysis indicated that the proteins that were upregulated during the 291 dry (breeding) season, such as ROPN1L, phospholipid hydroperoxide glutathione, HSPA2, and 292 SPA17, interacted with 10 other proteins. Among these interactions, only ROPN1L and 293 phospholipid hydroperoxide glutathione interacted with each other (Fig 2).  acrosomal HSP70 has a role in gamete interaction and fertilization [46], whereas HSP90AA1 expression has been correlated with the resistance of sperm to freezing [47,48] since this protein is 341 characterized as a ubiquitous molecular chaperone that provides protection and protein folding 342 during thermal stress and resistance against cell oxidative stress [49].

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HSPA2, which is a molecular chaperone that assists in the folding, transport, and assembly 344 of proteins in the cytoplasm, mitochondria, and endoplasmic reticulum and is a testis-specific 345 member of the 70-kDa family [50], has been suggested to be crucially involved in spermatogenesis 346 and meiosis [51]. In humans, the downregulation of HSPA2 mRNA was observed in testes with 347 abnormal spermatogenesis, and the protein expression was high in normal spermatogenesis and low 348 in spermatogenesis arrest [52]. Human HSPA2 was shown to regulate the expression of the sperm 349 surface receptors involved in sperm-oocyte recognition [53], and its depression in the testes was 350 also associated with spermatogenic impairment and the fertilization rate in men with azoospermia 351 who were treated with intracytoplasmic sperm injections [54]. complexes that generate the force for cilia motility and govern the beat frequency [56]. DNAH1 is 357 related to spermatogenesis and cell proliferation [57]. In humans, mutations in DNAH1 cause  In this context, Imai et al. [60] showed that the failure to express phospholipid 361 hydroperoxide glutathione peroxidase (GPX4) caused human male infertility, with 30% of men The acrosome, which is a membrane-bound exocytotic vesicle that is located over the 372 anterior portion of the nucleus, contains the hydrolytic enzymes that are required for the acrosome 373 reaction, binding of the zona pellucida (ZP), penetration through the ZP, and sperm-egg membrane 374 fusion, all of which are indispensable events during the fertilization process [62]. In the acrosome 375 membrane (internal and external membranes), the sperm acrosome membrane-associated family 376 (i.e., SPACA3, SPACA1, and SPACA4) [63,64] are sperm surface membrane proteins that may be 377 involved in the adhesion and fusion of the sperm to the egg prior to fertilization [65]. SPACA1 and 378 SPACA3 are localized in the acrosomal matrix, including the principal segment and equatorial 379 segment, and are proteins characterized as membrane antigens [63,65,66]. SPACA1 may be 380 involved in sperm fusion with the oölemma, since treatment of human sperm with the anti-SPACA1 381 antibody prevented sperm penetration into zona-free hamster eggs [63]. Fujihara et al. [67] 382 demonstrated that the SPACA1 protein was indispensable for the normal shaping of the sperm heads 19 383 during spermiogenesis in mice. In humans, this protein was identified as a sperm membrane antigen, 384 with a molecular mass ranging from 32 to 34 kDa [63]. in the egg or the spermatozoa have been found to be essential for fertilization.

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Until now, IZUMO1 has been found to be the essential protein on the sperm side for the 394 fusion process. As a testis-specific protein, IZUMO was discovered on the equatorial segment of the 395 acrosome-reacted mouse spermatozoa through proteomic analysis of the antigen recognized by the 396 monoclonal anti-mouse sperm antibody [70]. IZUMO is present in both mouse (~56 kDa protein) 397 and human (~38 kDa protein) sperm [71]. In mice, immunization with the IZUMO protein caused a 398 contraceptive effect in females, which was due to the significantly inhibited fusion of sperm to the 399 zona-free mouse eggs with the anti-PrimeB antibody. However, no effect on sperm motility was and morphology) were similar to those previously reported for fresh Amazon squirrel monkey 420 sperm (liquid fraction) and sperm from the coagulated fraction after dilution in ACP-118 [2][3][4]. 421 However, this was the first time that a comparison of these parameters during the dry and rainy 422 seasons was performed for this species.

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Although the seminal pH was higher in the dry season, it was slightly alkaline during both 424 seasons and similar to the range reported elsewhere for S. collinsi (pH 6.5-8.0) [2][3][4]  women, the acidic vaginal environment is toxic to sperm because the optimal pH for sperm viability 428 ranges from 7.0 to 8.5, and a reduction in sperm motility is seen at a pH of less than 6.0. However, 429 during human sexual intercourse, the vaginal epithelium produces a transudate that lubricates the 430 vagina and elevates the vaginal pH to 7.0 [82]. This physiological modification to accommodate the 431 alkaline pH of semen temporarily protects the spermatozoa and creates an optimal environment in 432 the cervix for sperm motility [83].