Spermatic RXFP2 expression levels and seminal INSL3 concentrations among beef bull ejaculates with different levels of sperm morphological normality

Abstract

The objective of this study was to examine the relationship between the insulin-like peptide 3 (INSL3) receptor (RXFP2) expression levels on spermatozoa and INSL3 concentrations in the seminal plasma of fresh semen from beef bulls with different levels of sperm morphological normality. Ejaculates (n = 44) were collected from 21 yearling Japanese Black beef bulls and categorized into three groups based on the levels of sperm morphological normality: High (normal morphology ≥ 80%; n = 23), Mid (< 80% & ≥ 65%; n = 10) and Low (< 65%; n = 11). Immunofluorescence was used to determine the localization and expression levels of RXFP2 in spermatozoa. Sperm RXFP2 was detected in the principal and equatorial segments of the acrosomal region, postacrosomal region, and neck in all groups. The levels of RXFP2 in the acrosomal principal segment, postacrosomal region, and neck in the Low group were significantly lower than those in the High and Mid groups, and those in the equatorial segment tended to be lower than those in the High group. The total level of RXFP2 in the Low group was also significantly reduced compared with that in the other two groups. Seminal plasma INSL3 concentrations were significantly higher in the Low group than in the other two groups, whereas testosterone levels did not differ significantly between the groups. In conclusion, RXFP2 levels were reduced in the sperm head and neck in bovine semen, with a lower level of sperm morphological normality, suggesting possible associations between increased sperm deformity and INSL3 receptor reduction. Higher seminal INSL3 concentrations in abnormal semen are probably related to fewer INSL3 receptors in spermatozoa.

Japanese Black (JB) cattle are the most common beef breed specific to Japan and are renowned for their exceptional meat quality, characterized by abundant marbling [1]. Almost all JB beef cattle are bred by artificial insemination (AI) with cryopreserved semen collected from a limited number of genetically superior beef bulls (sires) or embryo transfer [2]. Therefore, the economic value of JB beef sires is extraordinarily high. However, some candidate sires produce aberrant semen, including morphologically abnormal spermatozoa [3]. To address this problem, the general examination of semen characteristics includes the investigation of sperm morphological normality, although there have been fewer efforts to determine the causal factors of sperm deformity. Selection of Santa Gertudis beef bulls in USA with higher percentage (≥ 80%) of morphologically normal spermatozoa increased 5% of conception rates after the natural mating than unselected bulls, which 35% of bulls had lower percentage (< 79%) of morphologically normal spermatozoa [4]. Generally, inseminations of semen include 20–30% or more morphologically abnormal spermatozoa reduce pregnancy rates in cows and heifers [5, 6]. Thus, it has been considered that sperm morphological abnormality may detrimentally affect not only sperm motility and fertilization capacity but also sire conception rates [7,8,9]. In fact, the candidate of JB bulls which produce aberrant semen including morphologically abnormal spermatozoa are not selected as the sires in the AI center.

Recently, various new semen biomarkers for bulls’ infertility or subfertility have been reported [10,11,12]. Biomarkers include acrosomal, nuclear, and capacitation-related proteins in spermatozoa and seminal plasma proteins [10]. The detection or estimation of protein biomarker expression is superior to conventional methods, such as standard semen evaluation or testicular biopsy, because sperm function can be clarified noninvasively, especially in valuable sire candidates.

Insulin-like peptide 3 (INSL3) plays a crucial role in spermatogenesis in sexually mature rats and boars [13, 14]. Circulating INSL3 concentrations in peripubertal JB beef bulls producing abnormal semen containing high percentage (> 20%) of morphologically abnormal spermatozoa and poor sperm motility are lower than those in bulls with normal semen [3]. Relaxin family peptide receptor 2 (RXFP2), the cognate receptor for INSL3, was detected in the acrosomal equatorial segment of heads and the middle piece of flagella of frozen-thawed bull spermatozoa [15]. However, no studies have examined the relationship between sperm morphology, RXFP2 in fresh bovine spermatozoa, and INSL3 concentration in the seminal plasma of semen samples. Our previous study demonstrated higher levels of INSL3 in the preovulatory follicles of bovine ovaries [16]. Thus, the release of INSL3 into the oviduct upon ovulation could potentially modulate the function of sperm present in the oviduct awaiting fertilization.

In this study, we examined the relationship between RXFP2 expression levels in spermatozoa and INSL3 concentrations in the seminal plasma of fresh semen from JB beef bulls with lower levels of sperm morphological normality to discover any associations between spermatozoal shape just after ejaculation and the expression of molecules that may play roles in spermatogenesis and subsequent sperm maturation.

Materials and Methods

Preparation of samples

Ejaculates were collected by the technicians using an artificial vagina from sire candidates (JB bulls, > 1-year-old, 16.5 ± 0.4 month of age) for the purpose of routine examination of semen characteristics to evaluate their reproductive performance with the permission of the Hokubu Agricultural Technology Institute, Hyogo Prefectural Technology Center for Agriculture, Forestry and Fisheries (Hokubu Institute) under the research project plan “Improvement of Fertility Assay for Japanese Black Bull Spermatozoa (2016-2023).” They were used immediately for the subjective examination of sperm motility and then transported to our laboratory within 2.5 h at approximately 25–30°C for the further examination of sperm acrosomal proteins.

In this study, surpluses of ejaculates (44 samples from 21 bulls) were used after examination of semen characteristics. Of the surplus of each ejaculate, 2 ml semen underwent three washes with phosphate-buffered saline (PBS) containing 0.1% (wt/vol) polyvinyl alcohol (PVA; Sigma-Aldrich, St. Louis, MO, USA) through centrifugation at 700 × g for 5 min each time. Excess semen (1–2 ml) was centrifuged at 15,000 × g for 15 min at 4°C and the supernatant was retained and stored at –30°C for hormonal analysis. In addition, a general semen analysis was performed to ensure that both semen volume (3.98 ± 0.12 ml) and sperm concentration fell within the normal range (13.44 ± 0.61 billion/ml, mean value ± standard error).

Examination of sperm morphology

Aliquots of the washed sperm suspension were mixed with an equal volume of 0.25% (vol/vol) glutaraldehyde (Nacalai Tesque, Kyoto, Japan) in PBS, gently smeared onto glass slides, and air-dried. The samples were subsequently stained for 90 min in a phosphate-buffered Giemsa solution (Merck KGaA, Darmstadt, Germany) [17]. JPEG images for each preparation were obtained using a differential interference microscope with a glass objective (× 100), camera adaptor (U-TV0.5XC-3, Olympus, Tokyo, Japan), and microscopic digital camera. Approximately 100 Giemsa-stained spermatozoa were randomly observed in the images to calculate the percentage of spermatozoa with normal morphology, according to the criteria of a previous report [17]. Briefly, approximately 100 spermatozoa from each preparation were classified into five categories based on their morphology: sperm with normal morphology, sperm with aberrant heads, connecting pieces, middle pieces, and principal and end pieces. Based on the obtained results of the examination of sperm morphology, semen samples were classified into three groups: High (normal morphology ≥ 80%), Mid (< 80% & ≥ 65%) and Low (< 65%).

Hormone assays

The seminal INSL3 concentrations were measured using a time-resolved fluorescence immunoassay (TRFIA) established in the laboratory [3, 18, 19]. Briefly, microtitration plates (1244-550, PerkinElmer, Boston, MA, USA) were coated with 100 µl of anti-mouse IgG goat polyclonal antibody (01-18-06, Kirkegaard & Perry Laboratories, Gaithersburg, MD, USA) per well for 2 h at room temperature (RT). The wells were washed thrice with 300 µl of 0.15 M sodium chloride. Approximately 150 µl of DELFIA assay buffer (1244-111, PerkinElmer) was then added, and the plates were incubated at 4°C overnight. Prior to the assay, each seminal plasma sample was diluted (100 ×) using the DELFIA assay buffer. Immediately before the assay, the wells were drained, to which either 50 µl of synthetic bovine INSL3 [20] for the standards or 50 µl of diluted seminal plasma plus 50 µl of anti-bovine INSL3 mouse monoclonal antibody (2-8F) [20] was added to each well and incubated for 2 h. Subsequently, 50 µl of biotin-labeled canine INSL3 [21] was added, and the plates were incubated for 1 h at RT. The wells were then washed thrice with 300 µl TRFIA wash buffer (0.05 M Tris-HCl buffer containing 0.15 M sodium chloride and 0.05% Tween 20, pH 7.8), after which 100 µl of Eu-labeled streptavidin (1244-360, PerkinElmer) was added. The plates were then incubated for 30 min at RT. After washing thrice with 300 µl of wash buffer, 100 µl of enhancement solution (1244-105, PerkinElmer) was dispensed into each well, followed by shaking at 500 rpm for 15 min at RT. Finally, time-resolved fluorescence was measured using a Nivo Multimode Plate Reader (PerkinElmer). The minimum detection limit was 0.078 ng/ml and the reliable detection limit was 0.078–20 ng/ml. The intra- and inter-assay CVs were 3.5% (n = 4) and 18.6% (n = 40), respectively.

Seminal testosterone concentrations were measured using EIA, with an extraction procedure established in the laboratory [18, 21, 22]. The minimum detection limit was 0.078 ng/ml and the reliable detection limit was 0.078–20 ng/ml. The intra- and inter-assay CVs were 10.5% (n = 4) and 6.9% (n = 40), respectively.

Immunofluorescence staining of sperm RXFP2

Immunofluorescence for RXFP2 was performed as described previously [23, 24]. Ten µl of the suspension of washed spermatozoa (1.0 × 10⁸ cells/ml in PBS) was smeared onto a glass slide (Matsunami Glass Industries, Osaka, Japan) and air-dried. The sperm smears were treated with 4% (wt/vol) paraformaldehyde (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) for 20 min and washed twice with PBS. Each smear was permeated with 0.2% (vol/vol) Triton X-100 (Sigma-Aldrich) for 10 min. After washing twice, the smears were blocked with PBS containing 5% (wt/vol) bovine serum albumin (BSA, Sigma-Aldrich) was performed for 1 h at RT, and then the primary antibody (Anti-RXFP2 rabbit polyclonal antibody) (Cat. #GTX108235; GeneTex, CA, USA; 40 µg/ml in 5% BSA-PBS) was added and incubated overnight at 4°C. The slides were then washed twice with PBS. Alexa Fluor 488-labeled anti-rabbit IgG goat polyclonal antibody (Life Technologies, Carlsbad, CA, USA; 1:800 dilution in 5% BSA-PBS) was added as a secondary antibody and incubated for 1 h at RT in dark. Thereafter, the slides were washed twice with PBS and covered with Vectashield Mounting Medium (Vector Laboratories, Newark, CA, USA) and coverslips. The stained sperm smears were observed using an epifluorescence microscope (U-FBW mirror unit composed of a BP460-495 excitation filter, DM505 dichroic mirror, and BA510IF emission filter; Olympus), and approximately 100 spermatozoa per sample were photographed at an exposure time of 333.33 ms. Only spermatozoa in which the head was still attached to the tail were selected, ensuring that the sperm did not overlap with others. All experimental and imaging criteria were strictly maintained to minimize potential variations in fluorescence intensity. To verify reaction specificity. The control experiment involved inhibiting the primary antibody (working concentration: 40 µg/ml) by employing an excess of RXFP2 peptide (Cat. #AAP63831; Aviva Systems Biology, CA, USA) at a concentration of 60 µg/ml, surpassing the antibody concentration by 50-fold. The integrated density of RXFP2 in distinct regions of sperm was measured using ImageJ software (version 1.54 g; Rasband W. S., U.S. National Institutes of Health, Bethesda, MD, USA). A freehand selection tool was used to delineate each acrosome, equatorial segment, and postacrosomal region in the head or neck. The total RXFP2 level per spermatozoon was calculated by summing the counts of all above regions. This process was replicated using approximately 100 spermatozoa per ejaculate.

Data analysis

Analysis of variance (ANOVA) using the Generalized Linear Models procedure of SPSS version 26 software (IBM, Somers, Tokyo, Japan) was used to examine the differences among High-, Mid-, and Low-semen groups in sperm RXFP2 expression levels and seminal plasma hormone concentration. Post-hoc pairwise comparisons were performed using least significant differences (LSD). Pearson’s correlation coefficient values and their significance were determined between the rate of normal sperm morphology and seminal plasma INSL3 and testosterone concentrations using the correlate procedure in SPSS version 26. Data are expressed as means ± SEM. Differences were considered statistically significant at P < 0.05, and the tendency was 0.05 < P < 0.1.

Results

Percentage of morphologically normal spermatozoa in each group

The High (morphologically normal spermatozoa ≥ 80%), Mid (< 80% & ≥ 65%) and Low (< 65%) groups showed 88.3 ± 0.9% and 70.7 ± 1.3% and 49.9 ± 5.0% of morphologically normal spermatozoa, respectively. The High, Mid and Low groups showed the highest percentages of sperm deformity for head (5.8 ± 0.7%, 13.4 ± 2.7% and 33.9 ± 6.1%, respectively) among all the pieces (head, connecting, middle, principal and end). The percentages of morphologically normal and abnormal spermatozoa in each semen sample are shown in Supplementary Table 1. There was a significant effect of the group on the percentage of head deformities (P < 0.01). The Low group exhibited significantly higher percentages of head deformities than both the High and Mid groups (P < 0.01).

Distribution and levels of RXFP2 on spermatozoa

RXFP2 immunoreactivity was clearly observed in the acrosomal principal segment, acrosomal equatorial segment, postacrosomal region, and neck of spermatozoa in the High group (Fig. 1A and Fig. 2F) whereas the immunofluorescence intensities were weaker in the Low group (Figs. 1C and 2F). No RXFP2 immunoreactivity was observed in the spermatozoa of the High and Low groups when the anti-RXFP2 antibody pretreated with the blocking peptide was used (Fig. 1B and Fig. 1D>). Sperm morphological normality (High, Mid, and Low groups) affected the RXFP2 levels in the acrosomal principal segment, postacrosomal region in the head and neck, and total levels per sperm (P < 0.05). RXFP2 levels per spermatozoon in the acrosomal principal segment, postacrosomal region, and neck, as well as the total levels in the Low group, were lower than those in the High and Mid groups (P < 0.05) (Figs. 2A, C, D, E). The level of this receptor at the equatorial segment in the Low group tended to be lower than that in the other two groups (P = 0.08) (Fig. 2B). Moreover, bull age had no significant effect on total RXFP2 protein levels per spermatozoon (P = 0.726).

Fig. 1.

Representative images of immunofluorescence with the anti-RXFP2 antibody in the High (A) and Low (C) groups of sperm morphological normality in Japanese Black beef bulls. The images of immunofluorescence were merged with those of phase-contrast microscopy. Specific binding of the anti-RXFP2 antibody to sperm was confirmed by using the antibody pretreated with specific blocking peptide (B: High + blocking peptide, D: Low + blocking peptide).

Fig. 2.

Integrated densities (A: acrosomal principal segment, B: acrosomal equatorial segment, C: postacrosomal region, D: neck, E: head + neck) of the RXFP2 protein on head and neck parts in Japanese Black beef bulls with High (n = 23), Mid (n = 10), and Low (n = 11) groups of morphologically normal spermatozoa. Data of the integrated densities are presented as means ± SEM. The asterisk indicates a significant difference compared to the High and Mid groups (* P < 0.05). The pound denotes a differing tendency compared to the High and Mid groups (^# 0.05 < P < 0.1). Representative immunofluorescence images of High and Low groups were shown on the panel of F. In the image data, green fluorescence indicates RXFP2 protein, and each area for acrosomal principal segment (Acr), acrosomal equatorial segment (Equ), postacrosomal region (Post) and neck (Neck) are delineated by red, yellow, blue and orange lines, respectively.

Seminal INSL3 and testosterone concentrations

Sperm morphological normality (High, Mid and Low groups) affected seminal fluid INSL3 concentration (P = 0.002). In contrast, testosterone levels were not affected by sperm morphology. Seminal INSL3 concentrations were significantly higher in the Low group than in the High and Mid groups (P < 0.05) (Fig. 3A), whereas testosterone concentrations did not differ significantly among the groups (Fig. 3B). INSL3 concentrations in the seminal plasma were significantly negatively correlated with the percentage of sperm morphologically normal (P < 0.05) (Fig. 3C), whereas testosterone concentrations were not significantly correlated with sperm morphology (Fig. 3D). Moreover, bull age had no significant effect on seminal INSL3 or testosterone levels (P = 0.177 and P = 0.266, respectively).

Fig. 3.

Seminal plasma INSL3 (A) and testosterone (B) concentrations in Japanese Black beef bulls with High (n = 23), Mid (n = 10), and Low (n = 11) groups of morphologically normal spermatozoa and correlations of seminal INSL3 (C) and testosterone concentrations (D) with sperm morphology. Data are presented as means ± SEM. The asterisk indicates a significant difference compared to bulls with High and Mid groups of morphologically normal spermatozoa (* P < 0.05).

Discussion

Some JB beef bulls produce semen with a high percentage of morphologically abnormal spermatozoa; however, the underlying cause remains unknown [25]. The INSL3 receptor (RXFP2) is present in frozen-thawed bovine spermatozoa [15]. However, the distribution and levels of RXFP2 in spermatozoa in fresh semen and the relationship between sperm morphology and RXFP2 are yet to be elucidated. In the present study, we compared RXFP2 expression in spermatozoa among three levels of morphological normality in fresh semen from yearling JB beef bulls. As the results, the RXFP2 expression level was lower in the spermatozoa of abnormal semen with low percentage of morphologically normal spermatozoa (< 65%), especially at both of head and neck, compared to those of a high (≥ 80%) and intermediate (< 80% & ≥ 65%) levels of normal spermatozoa. These results suggest a possible link between increased sperm deformity and INSL3 receptor reduction in bovine semen. INSL3 promotes spermatogenesis in rats and boars [13, 14]. Mating cows or heifers with fresh or freeze-thawed semen showing higher percentages of deformed spermatozoa lowered conception rates [4,5,6]. Thus, the authors inferred that the RXFP2 expression level in spermatozoa in bovine ejaculates might reflect the morphological features that affect fertility in female cattle after AI. Although the causes of reduced RXFP2 in the spermatozoa of abnormal semen with higher malformation rates are unknown, the authors speculated that reduced INSL3 secretion, observed in the peripheral blood of bulls with abnormal semen [3], may be unable to upregulate its receptors in seminiferous tubule sperm. In this context, it has been suggested that physiological concentrations of LH upregulate LH receptors in rat Leydig cells in vivo and in vitro [26, 27].

In this study, we observed RXFP2 at the acrosomal principal segment, equatorial segment, postacrosomal region of the head, and at the neck in spermatozoa, whereas another research group detected the receptor only in the equatorial segment of the head and in the flagellar middle piece [15], even though the same antibody and cattle breed were used in these studies. A possible reason for the inconsistent results for receptor localization in spermatozoa might be that fresh semen was used in the current study, in contrast to frozen-thawed semen used in the previous study. It is well known that semen cryopreservation procedures impair sperm metabolism, motility, and membrane normality, reducing fertilization potential [28]. A direct comparison of RXFP2 expression in bovine spermatozoa between fresh and frozen-thawed semen also seems valuable because AI with frozen semen, not with fresh semen, is commonly practiced in JB cattle. It has been suggested that the intracellular second messenger in INSL3-RXFP2 signaling is cyclic adenosine monophosphate (cAMP) [29, 30]. Enhancement of intracellular cAMP signaling cascades is essential for bovine sperm capacitation [10] although the role of INSL3 in sperm function is unknown. Whether INSL3 plays a role in sperm capacitation and whether there are any differences in the effects of INSL3 between fresh and frozen-thawed semen remain to be determined.

In this study, we could not determine whether RXFP2 reduction caused sperm morphological abnormalities or vice versa. Some types of abnormal spermatozoa are difficult to identify by conventional light microscopy and their detection requires transmission electron microscopy [31]. In addition, advanced methodologies based on molecular biomarkers or capacitation-related events are emerging to detect functional defects in bovine spermatozoa with low fertility that cannot be diagnosed using conventional semen evaluation [10, 11, 32]. Therefore, RXFP2 immunostaining is a potential diagnostic method for detecting functional abnormalities in bovine spermatozoa.

INSL3 has been suggested as an indicator of endocrine function in the testes [30]. Plasma INSL3 concentrations were lower in the peripheral blood of JB beef bulls producing ejaculates with a higher percentage of malformed sperm and lower sperm motility than in bulls with normal semen [3]. In the current study, the authors found that semen with low rate of normal-formed spermatozoa (< 65%) revealed a higher INSL3 concentration in the seminal plasma compared to those with a high (≥ 80%) and intermediate (< 80% & ≥ 65%) rates. Furthermore, we observed a negative correlation between the percentage of normal spermatozoa and INSL3 concentrations in seminal plasma. These results suggest a possible association between higher seminal INSL3 levels and a lower number of INSL3 receptors distributed on the head and neck regions of spermatozoa in JB beef bulls. Further studies are required to elucidate the mechanisms through which these two phenomena are related. Higher INSL3 concentrations in the seminal plasma of bulls with more malformed spermatozoa observed in this study may provide practical value for seminal INSL3 as a fertility biomarker.

In the present study, seminal testosterone concentrations did not differ between bulls with normal semen and those with abnormal semen, whereas there was a clear difference in INSL3 levels, although the reason for this difference between both hormones, which are produced in the same Leydig cells, is unknown. Similarly, circulating INSL3 concentrations differed, while testosterone levels were similar between JB beef bulls with normal semen and ones with abnormal semen in a previous study [3]. This may be due to the highly fluctuating nature of blood testosterone levels, which are acutely responsive to pulsatile LH release from the pituitary gland, whereas INSL3 levels are comparatively stable [33]. A similar mechanism may explain the different results between INSL3 and testosterone in terms of testicular hormonal dynamics in seminal plasma.

In conclusion, the lower expression level of RXFP2 protein in the sperm head and neck in abnormal bovine semen with a higher percentage of malformation suggests possible links between increased sperm deformity and INSL3 receptor reduction. Seminal INSL3 concentrations negatively correlated with sperm morphological normality, whereas testosterone levels did not show such a correlation, highlighting the potential value of seminal INSL3 as a biomarker for assessing bull fertility.

Conflict of interests

The authors declare no conflicts of interest.